In previous work in our group (Havlin and Tycko, PNAS 2005), we carried out the first solid state NMR studies of protein folding. These studies focussed on the 35-residue protein domain HP35, which is known to contain three alpha-helical segments in its folded state and to be thermally stable up to approximately 70 C. HP35 was chosen because it can be readily synthesized by standard solid-phase peptide synthesis methods and because it has been the subject of numerous previous studies by experimental techniques and by computer modelling. In this work, we examined the dependence of solid state NMR signals from selectively isotopically-labeled HP35 on chemical denaturant (GdnHCl) concentration in frozen glycerol/water solutions (glass transition temperature of roughly -70 C). At low denaturant concentrations, 13C NMR signals characteristic of a helical protein were observed, as expected. At higher denaturant concentrations, two quite unexpected observations were made: (1) In the "fully unfolded" state (7 M GdnHCl), NMR signals from the three helical segments were markedly different, indicating a high level of disorder in the third helical segment, a mixture of residual helix content and disorder in the second helical segment, and an apparently low degree of disorder but no helix content in the first helical segment. This contradicts the simple assumption that the unfolded state is a uniform random coil; (2) Near the unfolding midpoint (4.5 M GdnHCl), the three helical segments appeared to have progressed to different stages along their respective unfolding paths, i.e., denaturation of HP35 cab not be described by a simple two-state model in which only the fully-folded and fully-unfolded states coexist. This work demonstrated that solid state NMR measurements can indeed provide new information about protein folding.[unreadable] [unreadable] In work that has been submitted for publication recently, we carried out solid state NMR measurements that directly probe backbone phi and psi torsion angles for a particular site (Valine-50) in the first helical segment. These measurements employ three techniques developed previously in our group, with abbreviated names CT-DQFD, DQCSA, and 2DEXMAS. When these techniques are applied to HP35 in its folded state, the data are fit best by a single conformation, very close to the helical phi and psi angles determined by crystallography for folded HP35. In the unfolded state (7 M GdnHCl), the combined data can not be fit by a single conformation. Instead, the data are well described by significant populations near two phi,psi pairs, namely -75,155 degrees and -115,75 degrees. The first of these (with approximately 33% of the population) corresponds to the polyproline II conformation that has been suggested by other groups to be a dominant conformation in unfolded proteins. The second (with approximately 67% of the population) is in the "transition region" between alpha-helical and beta-strand conformations, and was not anticipated. Interestingly, ab initio calculations of 13C chemical shifts indicate that these two conformations have similar chemical shifts, accounting for the observation of relatively sharp lines in solid state NMR spectra, which had suggested a high degree of structural order.[unreadable] [unreadable] Most recently, we have constructed an apparatus that permits rapid freezing of protein solutions for high temperatures, in approximately 10 microseconds. With this apparatus, we have carried out the first solid state NMR studies of a transient intermediate state in a protein folding process. Again using HP35, we find that rapid freezing from +90 C (above the thermal unfolding temperature) to -130 C (in cold isopentane liquid) traps an unexpected state of the protein, in which "unfolded" molecules coexist with "folded" molecules, but in which the "folded" molecules are in fact not fully folded. Instead, preliminary data suggests that the "folded" component has native-like secondary structure but incomplete formation of tertiary structure. Experiments of this type will continue in FY2009.